Saccharomyces cerevisiae possess a monofunctional, cytoplasmic NAD-dependent 5,10-methylenetetrahydrofolate (THF) dehydrogenase that converts 5,10-methylene-THF to 5,10-methenyl-THF (Barlowe, C. K., and Appling, D.R. (1990) Biochemistry 29, 7089-7094). We have now isolated the gene encoding this enzyme from a yeast genomic library using oligonucleotide probes based on internal peptide sequences from the purified protein. Nucleotide sequence analysis reveals a 320-amino acid open reading frame that contains both of the internal peptide sequences. The predicted molecular weight (36,236) is consistent with the estimated size (33,000-38,000) of the purified protein. Disruption of the chromosomal copy of the gene resulted in loss of NAD-dependent 5,10-methylene-THF dehydrogenase activity and led to a purine requirement in certain genetic backgrounds, confirming a role for this enzyme in the oxidation of cytoplasmic one-carbon units. A single gene was mapped to chromosome XI by hybridization to a yeast chromosomal blot. We propose MTD1 as the name for this gene. Northern analysis of total yeast RNA revealed a single transcript of approximately 1,100 nucleotides. Multiple transcription initiation sites were identified between 58 and 83 base pairs upstream of the start of translation. The amino acid sequences derived from the nucleic acid sequences of seven other methylene-THF dehydrogenases cloned to date have been found to be highly homologous. Although the predicted amino acid sequence of the yeast NAD-dependent enzyme shows slight homology to the other sequences, it appears to be only distantly related to the other 5,10-methylene-THF dehydrogenases.